Performance of cotton expressing Cry1Ac, Cry1F and Vip3Aa19 insecticidal proteins against Helicoverpa armigera, H. zea and their hybrid progeny, and evidence of reduced susceptibility of a field population of H. zea to Cry1 and Vip3Aa in Brazil

The genetically modified cotton DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 expressing Cry1Ac, Cry1F and Vip3Aa19 from Bacillus thuringiensis Berliner (Bt) has been cultivated in Brazil since the 2020/2021 season. Here, we assessed the performance of DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton expressing Cry1Ac, Cry1F and Vip3Aa19 against Helicoverpa armigera (Hübner), Helicoverpa zea (Boddie), and their hybrid progeny. We also carried out evaluations with DAS-21023-5 × DAS-24236-5 cotton containing Cry1Ac and Cry1F. In leaf-disk bioassays, DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 was effective in controlling neonates from laboratory colonies of H. armigera, H. zea and the hybrid progeny (71.9%–100% mortality). On floral bud bioassays using L2 larvae, H. zea presented complete mortality, whereas H. armigera and the hybrid progeny showed <55% mortality. On DAS-21023-5 × DAS-24236-5 cotton, the mortality of H. armigera on leaf-disk and floral buds ranged from 60% to 73%, whereas mortality of hybrids was <46%. This Bt cotton caused complete mortality of H. zea larvae from a laboratory colony in the early growth stages, but mortalities were <55% on advanced growth stages and on floral buds. In field studies conducted from 2014 to 2019, DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton was also effective at protecting plants against H. armigera. In contrast, a population of H. zea collected in western Bahia in 2021/2022 on Bt cotton expressing Cry1 and Vip3Aa proteins, showed 63% mortality after 30 d, with insects developing into fifth and sixth instars, on DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton. We conclude that H. armigera, H. zea, and their hybrid progeny can be managed with DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton; however we found the first evidence in Brazil of a significant reduction in the susceptibility to DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton of a population of H. zea collected from Bt cotton in Bahia in 2021/2022.


Introduction
Helicoverpa spp. is a group of harmful agricultural pests in several regions of the world. In Brazil, the most important Helicoverpa species are the Old World bollworm, Helicoverpa armigera (Hübner)-first recorded in 2013 in soybean in Goiás and Bahia states-, and the corn earworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) [1][2][3]. In the current Brazilian crop production systems, outbreaks of Helicoverpa species are favored by the overlapping and simultaneous cultivation of host plants, including soybean, maize, cotton, tomatoes, and dry bean crops [4]. Studies also reported the occurrence of interspecific hybridization between H. armigera and H. zea under laboratory conditions [5][6][7]. The successful hybridization in a restricted environment suggests that potential for multiple mating events in the field may provide opportunities to select well-adapted hybrid phenotypes [7]. Cases of resistance to Bt proteins in H. zea have been reported in the U.S. and China [8][9][10][11] and decreases in susceptibility to Cry proteins by H. armigera have been reported in U.S., India, and China [12,13].
In Brazil, H. armigera has great importance in soybean and cotton [1,4], while H. zea was most abundant in maize and cotton [14,15]. The adoption of genetically-modified (GM) plants expressing insecticidal proteins derived from the soil bacterium Bacillus thuringiensis Berliner (Bt) is the main management tactic used against Helicoverpa species in cotton, soybean and maize fields in Brazil [16,17]. The successful adoption of Bt crops has enabled more effective management of lepidopteran pests and reduced chemical insecticide applications in these crops worldwide [18][19][20].
In Brazil, Bt cotton was planted commercially for the first time in 2006. Transgenic cotton technologies are cultivated on approximately 1.4 million hectares (90% of the total area with Bt cotton) in recent cotton seasons [21]. The first generation of commercial Bt plants expressed single Bt proteins, but low compliance with resistance management strategies (e.g. refuge areas) in areas cultivated with Bt maize has favored the cross-crop resistance, affecting the performance of other Bt plants, including Bt cotton varieties [22]. The second and third generations of Bt cotton met the concept of 'gene pyramiding' with the expression of two or more Bt proteins with high toxicity and distinct modes of action to the same pest species [23,24]. For example, the DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 event (commercially named Wide-Strike 1 3) is part of the third generation of Bt cotton cultivated in Brazil and expresses Cry1Ac, Cry1F and Vip3Aa19 insecticidal proteins [25].

A. Description of pest species and hybridization crosses
The two Helicoverpa species, H. armigera and H. zea, were collected from the field and maintained at Corteva Agriscience insectary (Toledo, Paraná, Brazil). Larvae of H. zea were collected from corn ears and H. armigera from soybean plants in Brasilia, DF during the 2015 crop season. Insects were maintained on an artificial insect diet proposed by Greene et al. [26], being that new field-collected larvae from non-Bt maize (H. zea) and non-Bt cotton or non-Bt soybean (H. armigera) were introduced to the colonies every year (~500 larvae/year). The new field larvae were collected in Paraná, São Paulo or Goiás states from 2016 to 2019. Hereafter, we refer to these populations as Hz-Lab and Ha-Lab. To design hybridization crosses, adults were confined in a PVC cage (30 cm in diameter × 30 cm height). Only the crossing between (500 ♀ Ha-Lab × 500 ♂ Hz-Lab) produced eggs that produced neonates. Hybrid larvae (F 1 generation) were tested in confined laboratory conditions, to avoid any escapes to the environment.
In addition, during the 2021/2022 cotton season, a field population of H. zea was collected in Bt cotton expressing Cry1Ab, Cry2Ae and Vip3Aa (variety FM 985, FiberMax1 -BASF S. A., São Paulo, SP, Brazil) in Luis Eduardo Magalhães, BA (latitude 12˚07'29" S, longitude 460 8'28" W). After the collection, this population was transferred to an artificial diet [26] and maintained in a laboratory at Instituto Mato-Grossense do Algodão (IMAmt), Primavera do Leste, MT, Brazil. We refer to this population as Hz-field.
Before starting bioassays, all species were confirmed by PCR testing according to the methodology described by Perera et al. [27].
Bioassays were performed with leaf disks of Bt and non-Bt cotton varieties previously described from leaves excised of plants in vegetative (growth stage 15 -5th true leaf unfolded) and reproductive (growth stage 55 -squares distinctly enlarged) stages according to the phenological scale of Munger et al. [28]. Squares (bracts + buds) from a reproductive stage (growth stage 55) were also tested. Leaves and squares were removed from the upper third of the plants when they reached the respective phenological stage. Leaf disks 1.2 cm in diameter were cut using a metallic cutter. Leaf disks and squares were placed over a gelled mixture of water-agar 2.5% (1 ml/well) in 128-well bioassay trays (BIO-BA-128; CD International Inc., Pitman, NJ).
The vegetative and reproductive structures tested were separated from the water-agar layer by a filter paper disk. Then, a single neonate (Ha-Lab, Hz-Lab and their F 1 hybrid progeny) was placed on each leaf disk or a single L 2 larvae was placed on each square. The trays were sealed with self-adhesive plastic sheets (BIO-CV-16; CD International Inc.) that allowed for gas exchange and then placed in a climatic chamber (temperature 25 ± 2˚C, 60 ± 5% RH, and 14:10 h photoperiod). The experimental design was completely randomized with 8 replicates/ growth stage/cotton technology (each replicate was represented by 16 larvae). Mortality, damage on leaf disks and squares, and larval weight were recorded at 5 d after infestation. The damage on each leaf disk was recorded using two methods. We measured the area (cm 2 ) that was consumed by larva using a transparent grid ruler, and we also estimated the damage as percentage of the disk that was consumed. The damage on squares was classified as a surface feeding on the external side of the square, and as bored squares, when the larvae could feed inside the square. All live larvae in each replicate were pooled to obtain the weight of the replicate and recorded, since some larvae were too small to accurately weigh individually.

C. Survivorship of Helicoverpa species on cotton throughout the larval cycle
Leaves of Bt and non-Bt cotton varieties previously described were excised from greenhousegrown plants at the growth stage 15, according to the scale of Munger et al. [28]. In the laboratory, leaves were cut into pieces (6-8 cm 2 ) and placed on a gelled mixture of agar-water at 2.5% in 50-mL plastic cups. Then, a single neonate from the Ha-Lab or Hz-Lab populations was placed in each cup, with leaves replaced every 24 h. Cups were placed in a climatic chamber at 25 ± 2˚C, 60 ± 5% RH, and 14:10 h photoperiod. The bioassays with Ha-Lab and Hz-Lab populations were performed at Federal University of Santa Maria, RS, Brazil.
Similar bioassays were performed with the Hz-field population at Instituto Mato-Grossense do Algodão, Primavera do Leste, MT, Brazil. In bioassays, leaves were removed from cotton plants at the growth stage 15, cut into pieces and placed in 24-well plastic plates. Then, a single neonate (F 1 generation) was placed in each well. When larvae reach 3 rd instar, they were individualized in Petri dishes (10 cm diameter × 1.5 cm height), with leaves replaced daily, being maintained in the same environmental conditions previously described. The cotton varieties tested in bioassays using the Hz-field population were: 1) TMG 50WS3 (early Maturity Before bioassays, plants were checked for the presence of the expected Bt proteins using detection kits (Envirologix, QuickStix, São Paulo, SP, Brazil) for Cry1Ac, Cry1F and Vip3A proteins. The experimental design was completely randomized with 42 and 26 replicates of 10 neonates for Bt cotton treatments, respectively, and 13 replicates of 10 neonates for the non-Bt cotton in bioassays with Ha-Lab and Hz-Lab, whereas 5 replicates of 20 neonates/cotton variety was used in bioassays with Hz-field. For both species, larval survival was evaluated every 5 d.

D. Field efficacy of Bt cotton against artificial and natural infestations of H. armigera
Ten field experiments were conducted from 2014 to 2019 growing seasons across four different states in Brazil (Table 1). All trials were conducted followed strict adherence to Brazilian regulatory requirements in accredited and certified field research stations operated by Corteva Agriscience or SGS Company. Field trials were performed under regulated permits approved by the Comissão Técnica Nacional de Biossegurança (CTNBio). Treatments included were: 1) A Bt cotton variety containing events DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 (Wide-Strike1 3 Insect Protection) expressing Cry1Ac, Cry1F and Vip3Aa19 Bt proteins; 2) A Bt cotton variety containing events DAS-21023-5 × DAS-24236-5 (WideStrike1 Insect Protection) expressing Cry1Ac and Cry1F Bt proteins, and 3) A non-Bt isoline cotton variety with a similar genotypic background and belonging to the same maturity group as the Bt cotton varieties. The Bt cotton variety used was PHY 440 (Mid-full Maturity, PhytoGen 1 Cotton Seeds) in all treatments. Each field trial consisted of four replications for each treatment arranged in a randomized complete block design. Plot size varied among locations from 5 to 8 meters in length and 5 or 7 rows wide. Row spacing in all locations varied from 50 to 76 cm.
Field efficacy of Bt cotton against artificial infestations of H. armigera. For this study, only H. armigera was infested in the field trials because previous laboratory trials on leaf disks with H. zea showed complete mortality. Larvae of H. armigera were obtained from laboratoryreared colony maintained by Corteva Agriscience (Mogi Mirim Research Center, Mogi Mirim, São Paulo State, Brazil) or SGS Company (Piracicaba, SP, Brazil). Artificial infestations were conducted at two different cotton reproductive growth stages at all locations to ensure uniform pest pressure across experimental plots ( Table 1). The first artificial infestation was conducted at GS6: 65, at the beginning of flowering ("mid bloom"), and the second infestation at GS6: 65+, 10-12 d later [28]. For each plot, ten plants were randomly selected and each one was infested with 10 L1 larvae. Larvae were placed on the growing points of the selected plants, and then covered immediately with mesh cages (150 cm long × 50 cm wide × 150 cm high) to limit larval escape and to avoid mortality caused by natural enemies. Field evaluations consisted of recording the total number of cotton squares on 10 infested plants, the percentage of damaged squares, and the number of live larvae still present. These evaluations were performed 10 d after each infestation.
Field efficacy of Bt cotton against natural infestation of Helicoverpa spp. A natural infestation of Helicoverpa spp. occurred only in one trial at Rio Verde (GO) ( Table 1). At this location, plot inspections and evaluations were performed weekly. The data presented in this paper represent the sampling date when the peak number of damaged squares and the number of live larvae were recorded for the non-Bt treatment.

E. Data analysis
The number of insects tested and the number dead in leaf-disk and square bioassays, and survivorship of Ha-Lab, Hz-Lab and Hz-field throughout the larval cycle on Bt and non-Bt cotton leaves were used to estimate 95% confidence intervals (CIs) for the probability of mortality, according to a binomial distribution [29]. To perform these analyses, the function binom. probit from the package binom in R 3.1.0 [30] was used. Percent mortality rate on Bt and non-Bt cotton were declared significantly distinct if 95% CIs did not overlap [31,32]. The variables of larvae developing on Bt and non-Bt cotton technologies (consumed leaf area, percent of leaf consumption, bud damage, and larval weight) and data from field trials (artificial infestation) on the efficacy of Bt cotton technologies against H. armigera were analyzed using a linear mixed model (PROC MIXED procedure) and statistical significance were obtained by using Tukey's Honestly Significant Difference test with α = 0.05 in SAS [33]. Prior to analysis, data from each trial were evaluated to ascertain its validity by assessing the injury level in the non-Bt control. Trials selected for analysis only included those in which the non-Bt control averaged �10% square injury. Furthermore, prior to the combined analysis, each trial was analyzed individually and the mean square error (MSE) of the residual was used to evaluate the homogeneity of the variance error. Only trials that showed a ratio between the largest and smallest MSE �7 were included in the combined cross-trial analyses [34]. This procedure ensured that trials were homogeneous to avoid bias caused by differences among trials (sites and years). The data from the single, naturally-infested location of Helicoverpa spp. was subjected to t-test (PROC TTEST procedure) paired with α = 0.05 in SAS [33]. To improve the normal distribution, data of squares injured were log (x + 1) transformed, while data on number of larvae were transformed using ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi x þ 1 p . Non-transformed data are presented in figures.
Surviving neonates (on leaf-disk) and L2 larvae (on squares) of H. armigera (Ha-Lab) and F 1 hybrids on DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 and DAS-21023-5 × DAS-24236-5 cotton technologies had significant lower larval weights (<0.90 mg/larva on leaf-disk and <3.7mg/larva on floral buds) than on non-Bt cotton (>4.6 mg/larva on leaf-disk and >12.6 mg/larva on floral buds) (for each pest, growth stage and plant structure df (Num/Den) = 2/21; P < 0.0001). These results indicate that surviving larvae on Bt cotton technologies had more than 80% (on leaf-disk) and 70% (on squares) growth inhibition relative to those fed on non-Bt cotton. For H. zea (Hz-Lab), the larval weights were not computed due to the limited number of insects surviving on both Bt cotton technologies.

Discussion
The susceptibility of Helicoverpa species to Cry1F and Vip3Aa19 in Brazil have not been fully characterized; susceptibility to another Vip protein (e.g. Vip3Aa20) was reported for H. armigera and H. zea and to Cry1Ac for H. armigera, indicating that H. armigera was more tolerant to Vip3Aa20 than H. zea, but had high susceptibility to Cy1Ac [35,36]. To assess the performance of Cry1Ac, Cry1F and Vip3Aa19 Bt proteins expressed in DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton technology against the Heliothine species complex in Brazil, we performed laboratory and field studies with H. armigera and H. zea and the hybrid progeny of the species. In most experiments presented here, insects from laboratory colonies that were annually augmented with field-collected insects were used.
The relative high mortality levels observed for Helicoverpa species from laboratory-reared colonies and hybrids on leaves of DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton indicate that Cry1Ac, Cry1F and Vip3Aa19 are effective in protecting cotton plants against damage from these target pests. This 3-gene Bt cotton technology also improved protection against square damage caused by Helicoverpa species and hybrids compared to 2-gene Bt cotton and should improve yield protection, since larvae may prefer to attack reproductive structures of cotton [37,38]. According to Rios et al. [7], there is chance of interspecific crosses in Helicoverpa species, with interspecific crossing probably occurring when pest populations are at high densities, suggesting that populations of these pests should be maintained low levels in the field. Therefore, the effectiveness of DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton in Table 5 controlling Helicoverpa species may reduce population levels across the landscape, favoring that the 'species identities' will be maintained under field conditions. When comparing survival of H. zea colonies throughout the larval life cycle in two different studies (Tables 4 and 5), the field population of H. zea collected in Bt cotton expressing Cry1Ab, Cry2Ae and Vip3A in western Bahia, Brazil during the 2021/2022 season had lower mortality on DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton technology than was observed for the laboratory H. zea population. During this season, it was verified a high occurrence of larvae of the genus Helicoverpa spp. in the western Bahia, Brazil causing considerable damage in reproductive structures of Bt cotton expressing Cry1Ab, Cry2Ae and Vip3Aa [39]. These larvae were later identified by PCR tests as H. zea and was the source of Hz-field population here tested. Our findings suggest that the Hz-field population collected from a Bt cotton field expressing Cry1, Cry2 and Vip3 in Bahia has lower susceptibility to Cry1 and Vip3Aa in comparison to the Hz-Lab. While not a definitive test for resistance to the Bt proteins (a known laboratory-susceptible H. zea population was not tested in the same study as the Bahia-collected Hz-field population), the current evidence (collection of survivors in a Bt field and the laboratory bioassay findings relative to other findings with a H. zea laboratory population) implies that reduced control is possible in the field. Diminished susceptibility to Bt proteins in H. zea and H. armigera has been previously reported in several states of U.S., India, Pakistan, and China [8][9][10][11][12][13][40][41][42]. These observations reinforce the need for compliance with non-Bt refuge guidelines when planting Bt cotton technologies to retard the evolution of resistance in Helicoverpa species in Brazil.
Further investigation of Helicoverpa populations from the Bahia cotton-producing region are needed to fully understand the long-term implications for the durability of both Bt cotton and Bt maize against these species. For example, it will be important to confirm whether the Hz-field population is resistant to Cry1, Cry2 and/or Vip3Aa Bt proteins and to understand the performance of Hz-field on Bt maize hybrids. The ability of H. zea and H. armigera to produce hybrid offspring may also have implications for the development of resistance to Bt proteins in cotton-infesting populations of H. armigera that are not currently understood.
Currently, relevant exposure to Cry and Vip3Aa proteins by Helicoverpa species, which is part of DAS-21023-5 × DAS-24236-5 × SYN-IR102-7, is ongoing in Brazil, because of the widespread planting of Bt soybean, Bt cotton and Bt maize expressing a single-mode-of-action Bt proteins. Although H. armigera was first identified in soybean in Brazil [1], this species prefers cotton as a host plant [4]. Despite the relative high level of adoption of Bt cotton technologies expressing Cry proteins (~90% of the total cotton area), these technologies continue to provide reasonable levels of control against Helicoverpa species. The abundance of Helicoverpa species has not been quantified in cotton in Brazil, but studies stated a low abundance of H. armigera in soybean fields [20]. The low abundance of this pest in soybean is a positive aspect for the IPM, since same Bt proteins are expressed in cotton and soybean against H. armigera, indicating that broad adoption of Bt plants reduced population levels of this species.
From pest management perspective, the cotton technology DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 beyond reasonable levels of mortality against H. armigera, also reduced the damage to squares. These results expand those previously reported by Marques et al. [25], showing this same Bt cotton technology is highly effective against other lepidopteran pests of cotton. It is noteworthy that, the non-Vip3Aa containing products usually did not provide full control of S. frugiperda, and growers implemented multiple insecticide sprays that probably contributed to controlling Helicoverpa spp. as well. As growers are moving to Vip3Aa-containing varieties, less insecticide sprays are needed for lepidopteran control, reducing the monitoring of lepidopterans and increasing potential for escapes of Helicoverpa spp. in the field [39]. Therefore, to maintain the benefits of DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton for pest control in Brazil, where Helicoverpa species have numerous generations per year and are exposed to same mode-of-action Bt proteins in other crops, this GM cotton technology should be integrated with other control tactics, including the adoption of at least 20% of structured refuge (non-Bt cotton) for resistance management.